Project summary A fundamental gap in understanding exists between clinical observations on the importance of HSF1 levels in cancer and a complete knowledge of the mechanisms that drive HSF1 regulation. Tragically, breast cancer patients expressing both high HSF1 levels and high HSF1 activity exhibit a precipitous decrease in metastasis- free survival by 5 years (<15%) compared to patients expressing either high levels or high activity of HSF1 (~60%), or patients where neither HSF1 levels nor activity is high (>75%). HSF1 is the master regulator of the heat shock response that functions to maintain cellular protein folding homeostasis. While the regulation of HSF1 protein activity during acute stress has been heavily studied, little is known about the mechanisms that regulate HSF1 protein levels, or how HSF1 protein activity is dysregulated in cancer cells. The HSF1 gene is rarely mutated in cancer genomes, indicating that the aberrant levels of HSF1 in cancer are not driven by a mutation in the gene itself and must lie in another factor. We have recently identified a new regulator, the alternative splicing factor SF3B1, which controls both HSF1 protein levels and HSF1 protein activity. Intriguingly, gain-of-function mutations in SF3B1 are enriched in several cancers and small molecule inhibitors of SF3B1 have potent antitumor activity. Our long-term goal is to uncover the regulatory mechanisms connecting SF3B1 and HSF1, probe their involvement in breast cancer and other cancers, and exploit this pathway for the development of anticancer therapeutics. The overall objective of this proposal is to define both the direct and the indirect regulatory mechanisms connecting SF3B1 and HSF1. Our central hypothesis, based on our recent publication and preliminary data, is that SF3B1 simultaneously affects the cellular levels of the HSF1 protein and disables the negative feedback loops known to regulate its activity via distinct mechanisms. Completion of our first specific aim will reveal the steps in HSF1 gene expression sensitive to SF3B1, identify the necessary sequence elements and uncover candidate genes that mediate these effects. Our second specific aim will define a new regulatory pathway where SF3B1 indirectly influences HSF1 activity via the alternative splicing of an intermediary factor. Our approach is innovative because other HSF1 regulators are not known to affect both HSF1 levels and HSF1 activity. This proposed research is significant because it will enable new insights into HSF1 regulation that may be important for its role in normal growth, stress responses, development, and aging. Additionally, this research will provide a foundation for future work evaluating the role of the interaction between SF3B1 and HSF1 in breast cancer. Finally, this proposal will serve to significantly enhance exposure of students to biomedical research at the Florida Institute of Technology and strengthen the research infrastructure of the university, making it a perfect fit for an R15 AREA grant.